RNA Interference of Antifreeze Protein Gene in Tenebrio molitor Mediated by Bacterially Expressed dsRNA

Abstract

Abstract: In order to further study the non-freezing function of antifreeze protein（AFP）genes in Tenebrio molitor using RNAi technology, the RNAi fragment of Tmafp433 from T. molitor was inserted into L4440 dsRNA expression vector, and transformed into E. coli HT115（DE3）. The dsRNA corresponding to Tmafps, designated as Tmafp-dsRNA, was expressed by IPTG induction, and injected into the larvae after purification. The mRNA level of Tmafps was detected using real-time quantitative PCR. The results showed that the E. coli HT115（DE3）containing L4440-Tmafp recombinant plasmid can express Tmafp-dsRNA. After 24 h of injection, the expression of Tmafps in T. molitor was significantly decreased to 60.8% of the control, suggesting that the expression of Tmafps was inhibited by injecting the bacterially expressed Tmafp-dsRNA. The present results laid foundation for further research in afp gene function.

Key words: Tenebrio molitor, afp gene, RNA interference, Double-stranded RNA, L4440 vector

Shi Meng, Liu Xiaoning, Ma Ji. RNA Interference of Antifreeze Protein Gene in Tenebrio molitor Mediated by Bacterially Expressed dsRNA[J]. Biotechnology Bulletin, 2014, 0(8): 113-119.

References

［1］Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans［J］. Nature, 1998, 391（6669）：806-811.
［2］Lipardi C, Wei Q, Paterson BM. RNAi as random degradative PCR：siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs［J］. Cell, 2001, 107（3）：297-307.
［3］Yang G, You MS, Vasseur L, et al. Development of RNAi in insects and RNAi-based pest control ［M］//Stoytcheva M. Pesticides in the Modern World-Pests Control and Pesticides Exposure and Toxicity Assessment. Rijeka：InTech, 2011：28-38.
［4］Yu N, Christiaens O, Liu J, et al. Delivery of dsRNA for RNAi in ins-ects：an overview and future directions［J］. Insect Science, 2013, 20（1）：4-14.
［5］Baum JA, Bogaert T, Clinton W, et al. Control of coleopteran insect pests through RNA interference［J］. Nature Biotechnology, 2007, 25（11）：1322-1326.
［6］Liu S, Ding Z, Zhang CW, et al. Gene knockdown by intro-thoracic injection of double-stranded RNA in the brown planthopper, Nilapa-rvata lugens［J］. Insect Biochemistry and Molecular Biology, 2010, 40（9）：666-671.
［7］Luo Y, Wang X, Yu D, et al. Differential responses of migratory locu-sts to systemic RNA interference via double-stranded RNA injection and feeding［J］. Insect Molecular Biology, 2013, 22（5）：574-583.
［8］Zhu Q, Arakane Y, Beeman RW, et al. Functional specialization among insect chitinase family genes revealed by RNA interference
［J］. Proceedings of the National Academy of Sciences of the United States of America, 2008, 105（18）：6650-6655.
［9］王根洪, 祝慧敏, 罗会松, 等. 细菌表达dsRNA介导的家蚕FTZ-F1基因的RNA干扰［J］. 昆虫学报, 2011, 54（5）：596-601.
［10］Horwath KL, Easton CM, Poggioli GJ, et al. Tracking the profile of a specific antifreeze protein and its contribution to the thermal hysteresis activity in cold hardy insects［J］. European Journal of Entomology, 1996, 93：419-433.
［11］Qiu LM, Mao XF, Hou F, et al. A novel function-Thermal protective properties of an antifreeze protein from the summer desert beetle Microdera punctipennis［J］. Cryobiology, 2013, 66（1）：60-68.
［12］胡有燕, 牛娜, 迟胜起. 植物病毒源dsRNA原核表达条件的研究［J］. 青岛农业大学学报：自然科学版, 2011, 28（2）：103-106.
［13］张德咏, 朱春晖, 成飞雪, 等. 表达dsRNA的细菌提取液可抑制黄瓜花叶病毒对烟草的侵染［J］. 植物病理学报, 2008, 38（3）：304-311.
［14］Yang D, Lu H, Erickson JW. Evidence that processed small dsRNAs may mediate sequence-specific mRNA degradation during RNAi in Drosophila embryos［J］. Current Biology, 2000, 10（19）：1191-1200.
［15］Tuschl T, Zamore PD, Lehmann R, et al. Targeted mRNA degradation by double-stranded RNA in vitro［J］. Genes & Development, 1999, 13（24）：3191-3197.
［16］Duman JG, Horwath KL. The role of hemolymph proteins in the cold tolerance of insects［J］. Annual Review of Physiology, 1983, 45（1）：261-270.
［17］刘忠渊, 王芸, 吕国栋, 等. Tenebrio molitor抗冻蛋白基因家族cDNA 片段的克隆、序列分析及原核表达［J］. 遗传, 2006, 28（12）：1532-1540.
［18］Timmons L, Court DL, Fire A. Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans［J］. Gene, 2001, 263（1-2）：103-112.
［19］汪猷, 徐耀忠, 张伟君, 等. 应用³¹PN MR研究RNase A水解核糖核酸的机制［J］. 化学学报, 1988, 46（2）：204-205.
［20］Araujo RN, Santos A, Pinto FS, et al. RNA interference of the salivary gland nitrophorin 2 in the triatomine bug Rhodnius prolixus（Hemiptera：Reduviidae）by dsRNA ingestion or injection［J］. Insect Biochemistry and Molecular Biology, 2006, 36（9）：683-693.
［21］Knorr E, Vilcinskas A. Post-embryonic functions of HSP90 in Tribolium castaneum include the regulation of compound eye development［J］. Development Genes and Evolution, 2011, 221（5-6）：357-362.
［22］Alves AP, Lorenzen MD, Beeman RW, et al. RNA interference as a method for target-site screening in the western corn rootworm, Diabrotica virgifera virgifera［J］. Journal of Insect Science, 2010, 10（162）：1-16.
［23］Wang L. Identification and Characterization of Protein Enhancers of Antifreeze Proteins from Overwintering Beetle Larvae Dendroides Canadensis［M］. Pro Quest, 2005.
［24］Hou F, Ma J, Liu XN, et al．Seasonal changes in antifreeze protein
gene transcription and water content of bettle Microdera punctipen-nis（Coleoptera：Tenebrionidae）from Gurbantonggut desert in central Asia［J］. Cryo Letters, 2010, 31（5）：359-370.
［25］王晶, 邱立明, 王岩, 等. 光滑鳖甲Anatolica polita抗冻蛋白基因的发育阶段性表达以及低温对其表达的影响［J］. 新疆农业科学, 2010, 47（9）：1697-1702.
［26］Andorfer CA, Duman JG. Isolation and characterization of cDNA clones encoding antifreeze proteins of the pyrochroid beetle Dendroides canadensis［J］. Journal of Insect Physiology, 2000, 46（3）：365-372.

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